Nuclear targeting sequence

ABSTRACT

The present provides nuclear localization signaling (NLS) sequences derived from titin, comprised of amino acids 181-220: SVGRATSTAE LLVQGEEEVP AKKTKTIVST AQISESRQTR and fragments thereof, such as amino acids 193-208: VQGEEEVP AKKTKTIV; amino acids 199-208: VPAKKTKTIV; and amino acids 200-206: PAKKTKT. The NLS sequences can be linked to agents, such as peptides, proteins or nucleotides, for transporting the agents into the nucleus of cells, and the NLS-agent complex can be further linked to antibodies or ligands for specific binding to cells. Also provided is a method for constructing cDNAs comprising combining a NLS sequence with a nucleic acid sequence for a target protein for expression and entry of the target protein into the nucleus of cells, which then can perform specific functions therein.

The present application claims priority to U.S. Provisional ApplicationNo. 60/633,243, filed Dec. 3, 2004, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the translocation of proteinsand other compounds into and out of the nucleus of cells and, moreparticularly, relates to novel nuclear localization signal sequences andnuclear export signal sequences and their uses, such as, for example andwithout limitation, regulation of nucleic acid expression, transfectionof eukaryotic cells, gene therapy, protection from toxic chemicals,transport of anti-cancer agents, etc.

2. Description of Related Art

The exchange of macromolecules between the cytoplasm and the cellnucleus is a basic biological process in eukaryotic cells central to theregulation of gene expression (which underlies all aspects ofdevelopment, morphogenesis, and signaling pathways in eukaryoticorganisms). Nuclear traffic occurs exclusively through the nuclear porecomplex (NPC), a huge multi-proteic complex which lies across thenuclear membrane. While small molecules (up to 40-60 kDa) can diffusethrough the NPC, nuclear import of larger molecules, such as proteins,is mediated by specific nuclear localization signal (NLS) sequencescontained either in the transported molecule (Garcia-Bustos et al.,Biochim. Biophys. Acta 1071:83-101, 1991) or contained in a shuttleprotein which binds to the protein being transported.

NLS sequences typically are small, mostly basic, amino acid sequenceswhich can be classified into three general groups: (i) a monopartite NLSexemplified by the SV40 large T antigen NLS (PKKKRKV); (ii) a bipartitemotif consisting of two basic domains separated by a variable number ofspacer amino acids and exemplified by the Xenopus nucleoplasmin NLS(KRXXXXXXXXXXKKKL); and (iii) noncanonical sequences such as M9 of thehnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeastGa14 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481,1991).

The steps involved in the import mechanism of proteins into eukaryoticnuclei have been elucidated (Nigg, E. A., Nature, 386:779-87, 1997;Gorlich, D., EMBO J., 17:2721-7, 1998). To be transported, the NLSsequence is recognized by members of the importin family of proteins(also referred to as karyopherins), which then act as carriers totransport the substrate protein across the NPC. Inside the nucleus, theimportin-substrate complex dissociates, liberating the substrateprotein, and the importin carrier ultimately returns to the cytoplasm.The small GTPase Ran plays a pivotal role in this process by promoting,in its GTP-bound form, the dissociation of the import complex and thesubsequent recycling of the importin carrier.

Once in the nucleus, many proteins are transported back to the cytoplasmas an essential step in their biological function. The export ofmacromolecules from the nucleus also relies on the existence of aspecific signal in the substrate to be exported. For example, the Revprotein of human immunodeficiency virus type 1 (HIV-1) exits thenucleus, facilitating export of the unspliced viral RNA (Pollard andMalim, Ann. Rev. Microbiol., 52:491-532, 1998). Rev protein nuclearexport is mediated by a specific nuclear export signal (NES) sequenceconsisting of the leucine-rich sequence, LPPLERLTL, found also inproteins of other viruses (Dobbelstein et al., EMBO J. 16:4276-4284,1997). Additionally, numerous cellular proteins, such as I-KB and MAPKK,contain potential NES sequences that may regulate the biologicalactivity of these proteins by controlling their nuclear export (Ullmanet al., Cell 90:967-970, 1997). Known NES sequences essentially areshort, leucine-rich, hydrophobic peptide motifs which mediate thehandling of the substrate by other members of the importin β family ofproteins, called exportins. Nuclear import and export processes thus aretightly linked.

The relatively small size of the NLS and NES sequences and, moreimportantly, the lack of clear and consistent consensus motifs in thesesignals, make it difficult to predict their presence in a given proteinbased solely on the analysis of its amino acid sequence. Furthermore,even if a consensus NLS or NES is found, it may not represent afunctional signal. For example, β-glucuronidase (GUS), a commonly-usedreporter enzyme which resides exclusively in the cell cytoplasm, carriesa perfect, albeit non-functional, bipartite NLS sequence at its carboxyterminus. The only practical way to identify active NLS or NES sequencesis by microinjecting (Guralnick et al., Plant Cell 8:363-373, 1996) orexpressing the protein of interest in eukaryotic cells (Varagona et al.,Plant Cell 3:105-113, 1991), heterokaryon formation (Michael et al.,Cell 83:415-422, 1995), or using an in vitro transport system(Ossareh-Nazari et al., Science, 278:141-144, 1997).

A need exists, therefore, for determining new and unique NLS sequenceswhich can translocate proteins and other compounds effectively andefficiently into or out of the cell nucleus.

SUMMARY OF THE INVENTION

The present invention fulfills this need by providing nuclearlocalization signaling (NLS) sequences derived from titin, a largemuscle protein, comprised of amino acids 181-220: SVGRATSTAE LLVQGEEEVPAKKTKTIVST AQISESRQTR (SEQ ID NO: 1) and fragments thereof.

Fragments of the NLS sequences derived from titin include, withoutlimitation, amino acids 193-208: VQGEEEVP AKKTKTIV (SEQ ID NO: 2); aminoacids 199-208: VPAKKTKTIV (SEQ ID NO: 3); or amino acids 200-206:PAKKTKT (SEQ ID NO: 4).

The NLS sequences can be linked to one or more agents, such as peptides,proteins or nucleotides, in order to transport the agents into thenucleus of mammalian, preferably human, cells.

In an embodiment of the present invention, the NLS sequences linked toone or more agents also can be linked to binding reagents, such asantibodies or ligands, to form an NLS-agent-antibody or NLS-agent-ligandcomplex, which is capable of binding to specific cell surface-expressingantigens or receptors, respectively, on the plasma membranes of cells.The complexes then enter into the cytoplasm of the cells by endocytosis,after which they are transported into the nucleus of the cells throughan importin-NLS pathway.

In another embodiment, a method is provided for constructing cDNAscomprising combining an NLS sequence of the present invention with anucleic acid sequence for a target protein in order to provide amechanism for expression and entry of the target protein into thenucleus of cells, which then can perform one or more specific functions,such as, without limitation, protecting the nucleus from toxicchemicals, radiation or other DNA-modifying agents; regulation oftranscription, development or differentiation; induction of DNA arrest(blockade); apoptosis (cell death) or DNA synthesis; delivery ofanti-cancer agents to rapidly dividing cells, or any procedure where anagent is to be localized to the nucleus of a target cell for anypurpose.

In a further embodiment, kits are provided which contain an NLS sequenceof the present invention combined with a cDNA or RNA construct to forman NLS-cDNA or NLS-RNA construct in order to transfect DNA or RNA ofcells. The NLS-cDNA or NLS-RNA constructs are targeted to the nucleus ofa cell where they are incorporated into the genome of the cell and thenexpressed as a mRNA, ultimately to be translated into one or moreproteins. Other NLS-cDNA or NLS-RNA constructs can transfer silencingRNA into the nucleus of a cell in order to interfere with transcriptionof a native mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the transient expression of pEGFP-C1 constructs infour different cell lines. Stars indicate nuclear localization andarrows indicate cytoplasmic localization;

FIG. 2 illustrates intracellular localization of titin N- and C-terminusfragments in COS-7 cells;

FIG. 3 is a design of subclones of human titin N-terminus for searchingpotential nuclear localization signal (NLS) sequences;

FIG. 4 illustrates the intracellular localization of subclones of humantitin N-terminus in COS-7 cells;

FIG. 5 illustrates localization of subclones of human titin N-termini inMG63 cells;

Table 1 lists the primers used to make the NLS-localizing constructs inpEGFP-C1; and

Table 2 provides the predicted NES sequences in human titin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides for the first time nuclear localizationsignaling (NLS) sequences derived from the large muscle protein, titin,comprised of amino acids 181-220: SVGRATSTAE LLVQGEEEVP AKKTKTIVSTAQISESRQTR (SEQ ID NO: 1) and fragments thereof. Fragments of the NLSsequences derived from the titin protein include, without limitation,amino acids 193-208: VQGEEEVP AKKTKTIV (SEQ ID NO: 2); amino acids199-208: VPAKKTKTIV (SEQ ID NO: 3); or amino acids 200-206: PAKKTKT (SEQID NO: 4).

The NLS sequences can be linked to agents, such as peptides, proteins ornucleotides, in order to transport the agents into the nucleus ofmammalian, preferably human, cells.

In an embodiment of the present invention, the NLS sequences linked toone or more agents also can be linked to binding reagents, such asantibodies or ligands, to form an NLS-agent-antibody or NLS-agent-ligandcomplex, which is capable of binding to specific cell surface-expressingantigens or receptors, respectively, on the plasma membranes of cells.The complexes then enter into the cytoplasm of the cells by endocytosis,after which they are transported into the nucleus of the cells throughan importin-NLS pathway.

In another embodiment, a method is provided for constructing cDNAs,comprised of combining a NLS sequence of the present invention with anucleic acid sequence for a target protein in order to provide amechanism for expression and entry of the target protein into thenucleus of cells, which then can perform one or more specific functions,such as, without limitation, protecting the nucleus from toxicchemicals, radiation or other DNA-modifying agents; regulation oftranscription, development or differentiation; induction of DNA arrest(blockade); apoptosis (cell death) or DNA synthesis; delivery ofanti-cancer agents to rapidly dividing cells, or any procedure where anagent is to be localized to the nucleus of a target cell for anypurpose.

In a further embodiment, kits are provided which contain an NLS sequenceof the present invention combined with a cDNA or RNA construct to forman NLS-cDNA or NLS-RNA construct in order to transfect DNA or RNA ofcells. The NLS-cDNA or NLS-RNA constructs can be targeted to the nucleusof a cell where they are incorporated into the genome of the cell andthen expressed as a mRNA, ultimately to be translated into one or moreproteins. Other NLS-cDNA or NLS-RNA constructs can transfer silencingRNA into the nucleus of a cell in order to interfere with transcriptionof a native mRNA.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably and refer to any polymer of amino acids (dipeptideor greater) linked through peptide bonds. Thus, the terms “peptide,”.“polypeptide,” and “protein” include oligopeptides, protein fragments,analogues, nuteins, fusion proteins and the like.

As used herein, the terms “transfect,” “transfection” or “transfecting”is meant to indicate the act or method of introducing a molecule, suchas a nucleic acid or other compositions of the present inventionincluding, but not limited to, peptides and proteins.

Meanings of the term “gene expression” are known to those with skill inthe art. “Gene expression” includes the production of a protein from RNAor DNA and production of an RNA from a DNA. A gene is said to be“expressed” when it is transcribed into RNA, but this meaning alsoincludes translation into a peptide or protein. The term “geneexpression” is often shortened to “expression,” “expressed,” or thelike. Additional meanings of the term “gene expression” are known tothose with skill in the art.

The term “binding reagent” and like terms refer to any compound,composition or molecule capable of specifically or substantiallyspecifically (that is with limited cross-reactivity) binding anothercompound or molecule, which, in the case of immune-recognition, is anepitope. A “binding reagent type” is a binding reagent or populationthereof having a single specificity. The binding reagents typically areantibodies, preferably monoclonal antibodies, or derivatives or analogsthereof, but also include, without limitation: Fv fragments; singlechain Fv (scFv) fragments; Fab′ fragments; F(ab′)2 fragments; humanizedantibodies and antibody fragments; camelized antibodies and antibodyfragments; and multivalent versions of the foregoing. Multivalentbinding reagents also may be used, as appropriate, including withoutlimitation: monospecific or bispecific antibodies, such as disulfidestabilized Fv fragments, scFv tandems ((scFv)2 fragments), diabodies,tribodies or tetrabodies, which typically are covalently linked orotherwise stabilized (i.e., leucine zipper or helix stabilized) scFvfragments. “Binding reagents” also can include aptamers, as aredescribed in the art, as well as ligands that bind specific receptormoieties found on plasma membranes of cells.

Methods of making antigen-specific binding reagents, includingantibodies and their derivatives and analogs and aptamers, arewell-known in the art. Polyclonal antibodies to specific plasma membraneantigens can be generated by immunization of an animal. Monoclonalantibodies can be prepared according to standard (hybridoma)methodology. Antibody derivatives and analogs, including humanizedantibodies can be prepared recombinantly by isolating a DNA fragmentfrom DNA encoding a monoclonal antibody and subcloning the appropriate Vregions into an appropriate expression vector according to standardmethods. Phage display and aptamer technology is described in theliterature and permit in vitro clonal amplification of antigen-specificbinding reagents with very affinity low cross-reactivity. Phage displayreagents and systems are available commercially, and include theRecombinant Phage Antibody System (RPAS), commercially available fromAmersham Pharmacia Biotech, Inc. of Piscataway, N.J., and the pSKANPhagemid Display System, commercially available from MoBiTec, LLC ofMarco Island, Fla. Aptamer technology is described, for example andwithout limitation, in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,840,867and 6,544,776.

The NLS-nucleic acid-antibody or ligand constructs of the presentinvention can be formulated using a variety of noncovalent and covalentapproaches to associate the NLS sequences to the nucleic acids, whichthen are associated with an antibody or ligand specific for an antigenor receptor, respectively, of a particular cell plasma membrane. Asuitable protocol for the NLS-nucleic acid-antibody or ligand constructsof the present invention includes, for example, mixing the NLS sequencewith a nucleic acid, such as cDNA or RNA, to form ionic complexesbetween the negatively charged DNA, the NLS sequence and the antibody orligand. These complexes then can be used to transfect cells usingconventional methods well known by those skilled in the art. Significantenhancement of transgene expression can be achieved following directmicroinjection of the preformed NLS-nucleic acid-antibody or ligandconstructs of the present invention into the cytoplasm of cells. Anothersuitable protocol for formulating the NLS-nucleic acid-antibody orligand constructs of the present invention includes, for example, fusingor convalently binding the NLS sequence to a cationic moiety such aspoly/oligolysine or a histone 1-DNA-binding domain, which then iscovalently bonded to the antibody or ligand. Still another protocol forformulating the NLS-nucleic acid-antibody or ligand constructs of thepresent invention includes, for example, coupling the NLS sequences to anucleic acid, such as cDNA or RNA, by chemically conjugating the NLSsequence to the nucleic acid, which then is chemically conjugated to theantibody or ligand. Crosslinking agents can include, for example andwithout limitation, cyclo-propapyrroloindole and4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimideester, together with photoactive p-azido-tetrafluoro-benzyl-NLS sequenceconjugates. A further protocol for synthesizing the NLS-nucleicacid-antibody or ligand constructs of the present invention includes,for example, coupling streptavidin-conjugated NLS sequences tobiotinylated nucleic acids, such as cDNA or RNA and biotinylatedantibodies or ligands.

Any nucleic acid can be used in combination with the present invention.A useful nucleic acid encompasses any naturally occurring or syntheticnucleic acid, polynucleotide, derivative, or analogue thereof. One withordinary skill in the art will be able to determine which nucleic acidis useful and will be able to construct the useful nucleic acid.Sequence information for many useful nucleic acids is available indatabases known to one with skill in the art (see for example, McKusick,Mendelian Inheritance in Man. Catalogs of Human Genes and GeneticDisorders. Baltimore: Johns Hopkins University Press (1998, 12th ed.);and Online Mendelian Inheritance in Man, OMIM Center for MedicalGenetics, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.) (1999) World Wide Web URL: “http://www.ncbi.nlm.nih.gov/omim”).Alternatively, many useful nucleic acids are commercially available. Inaddition, one with ordinary skill in the art is able to determine thesequence of a region of nucleic acid using methods known in the art.

The nucleic acid can be, but is not limited to, single-stranded nucleicacid, double-stranded nucleic acid, polynucleotides, DNA, RNA, andsingle- or double-stranded viral nucleic acid. In certain preferredembodiments, the nucleic acid comprises an expression vector. Theexpression vector can include a gene sequence under the control of apromoter region which one with skill in the art can design to becompatible with a host cell, such that, the gene is expressed aftertransfer into the nucleus. In certain embodiments, the nucleic acid canbe a plasmid, a circular nucleic acid, a linear nucleic acid, a viralvector, a therapeutic vector, and the like.

In certain embodiments, the nucleic acid comprises an antisense nucleicacid targeted to complementary sequences in the nucleus. Specifically,the bridging of introns, exons, and intron-exon boundaries iscontemplated with antisense strands or antisense encoding vectors. Incertain embodiments, the nucleic acid encodes an expression product,wherein the expression product comprises a peptide, a polypeptide, aprotein, a fusion protein, or an antisense nucleic acid. In certainembodiments, the NLS-cDNA or NLS-RNA has an antisense activity, whereinthe antisense activity is localized in the nucleus, not in thecytoplasm, because the NLS directs the complex to the nucleus andretains it in the nucleus. In certain embodiments wherein an antisensenucleic acid is expressed from a nucleic acid annealed to an NLS-cDNA orNLS-RNA, the antisense reaction can take place in any compartment of acell or even outside of a cell. In certain embodiments, the nucleic acidencodes a gene, a reporter gene, a gene fusion, a transgene, or atherapeutic gene.

In certain embodiments, the nucleic acid is a therapeutic vector(including plasmid, expression, viral, and the like) capable ofexpressing a therapeutic gene in the host cell (the cell into which thetherapeutic vector is transferred). It is preferred that the therapeuticvector express the therapeutic gene product either constitutively orinducibly in the host cell. The NLS-cDNA or NLS-RNA can be annealed tothe non-coding strand of a promoter having control over the expressionof the gene product, wherein such design stimulates expression. Thetherapeutic vector can comprise a DNA vaccine.

Many desirable vectors, plasmids, expression vectors, DNAs, RNAs,oligonucleotides, strands of nucleic acid, and the like are readilyavailable through commercial sources and are useful in certainembodiments of the present invention as a nucleic acid (e.g., Roche,Stratagene, In Vitrogene, Promega, PerSeptive Biosystems, ResearchGenetics, and the like). Additionally, a nucleic acid can be produced bytechniques of molecular biology known to those of ordinary skill in theart (see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press). The meaningof terms such as “expression vector,” “vector,” “expression construct,”or “construct” that are used in certain embodiments are known to thoseof ordinary skill in the art. The terms “expression vector,” “vector,”“expression construct,” or “construct” are used interchangeably and, ingeneral, refer to any nucleic acid that encodes an expression product.The terms “expression vector,” “vector,” “expression construct,” or“construct” also are known to one with skill in the art. In certainembodiments, the nucleic acid is expressed. In certain preferredembodiments, the resulting transcript may be translated into a protein,but it need not be. Thus, in certain embodiments, expression includesboth transcription of a gene and translation of an RNA into a geneproduct.

Particularly useful vectors are contemplated to be those vectors inwhich a coding portion of the DNA segment, whether encoding a fulllength protein, polypeptide or smaller peptide, is positioned under thetranscriptional control of a promoter. In certain aspects “promoter”refers to a DNA sequence recognized by the synthetic machinery of thecell, or introduced synthetic machinery, required to initiate thespecific transcription of a gene.

The promoter may be in the form of the promoter that is naturallyassociated with a gene, as may be obtained by isolating the 5′non-coding sequences located upstream of the coding segment or exon, forexample, using recombinant cloning and/or PCR™ technology

In certain embodiments, the particular promoter that is employed tocontrol the expression of a nucleic acid is not believed to be critical,so long as it is capable of expressing the nucleic acid in the targetedcell. Thus, where a human cell is targeted, it is preferable to positionthe nucleic acid coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell. Generallyspeaking, such a promoter might include either a human or viralpromoter. The selection and use of such particular promoters will beapparent to those with skill in the art (see, e.g., U.S. Pat. No.5,858,774 to Malbon et al., incorporated herein by reference;Gene-Expression Systems (1998) Fernandez et al., eds. Academic Press; M.Kriegler, Gene Transfer and Expression: A Laboratory Manual (1991)Oxford University Press; and Gene Expression: General and Cell TypeSpecific (1993) M. Karin (ed.) Birkhauser).

The ability to specifically inhibit gene function in a variety oforganisms utilizing antisense RNA or dsRNA-mediated interference (RNAior dsRNA) is well-known in the field of molecular biology (see forexample C. P. Hunter, 1999, Current Biology, 9:R440-442; Hamilton etal., 1999, Science, 286:950-952; and S. W. Ding, 2000, Current Opinionsin Biotechnology, 11:152-156, hereby incorporated by reference in theirentireties). Interfering RNA, either double-stranded interfering RNA(dsRNAi or dsRNA) or RNA-mediated interference (RNAi), typicallycomprises a polynucleotide sequence identical or homologous to a targetgene, or fragment of a gene, linked directly, or indirectly, to apolynucleotide sequence complementary to the sequence of the target geneor fragment thereof. The dsRNAi may comprise a polynucleotide linkersequence of sufficient length to allow for the two polynucleotidesequences to fold over and hybridize to each other, although a linkersequence is not necessary. The linker sequence is designed to separatethe antisense and sense strands of RNAi significantly enough to limitthe effects of steric hindrance and allow for the formation of dsRNAimolecules and should not hybridize with sequences within the hybridizingportions of the dsRNAi molecule. The specificity of this gene silencingmechanism appears to be extremely high, blocking expression only oftargeted genes, while leaving other genes unaffected.

Accordingly, one method for disrupting a targeted gene according to thepresent invention includes associating a NLS sequence either to a dsRNAor RNAi, wherein the dsRNA or RNAi is comprised of polynucleotidesequences identical or homologous to the targeted gene or a homologuethereof. The terms “dsRNAi,” “RNAi,” and “siRNA” are usedinterchangeably herein unless otherwise noted.

RNA containing a nucleotide sequence identical to a fragment of thetarget gene is preferred for disruption; however, RNA sequences withinsertions, deletions, and point mutations relative to the targetsequence also can be used for inhibition. Sequence identity may beoptimized by sequence comparison and alignment algorithms known in theart (see Gribskov and Devereux, Sequence Analysis Primer, StocktonPress, 1991, and references cited therein) and then calculating thepercent difference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Alternatively, the duplex region of the RNA may bedefined functionally as a nucleotide sequence that is capable ofhybridizing with a fragment of the target gene transcript.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand(s);the promoters may be known inducible promoters, such as baculovirus. TheRNA strands may or may not be polyadenylated; the RNA strands may or maynot be capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). The use and production of an expression construct areknown in the art (see for example, WO 97/32016; U.S. Pat. Nos.5,593,874; 5,698,425; 5,712,135; 5,789,214; and 5,804,693; and thereferences cited therein). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof. Alternatively, the RNA may be used with no, ora minimum of, purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

Preferably, and most conveniently, dsRNAi can be targeted to an entirepolynucleotide sequence of the targeted gene.

The present invention is more particularly described in the followingnon-limiting example, which is intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLE Introduction

Titin is a giant protein expressed in cardiac, skeletal and smoothmuscle tissues which is responsible for muscle elasticity and forproviding a scaffold for assembly of sarcomeric proteins. The fulllength sequence of the titin gene contains 363 exons which encode a4,200 kDa protein having 38,138 amino acid residues. The titin proteinmainly is composed of immunoglobulin (Ig), fibronectin III (Fn-III)domains and PEVK repeats (a ˜28 residue, P, E, V, K-enriched motif),which contribute to the elasticity of the titin protein. Differentcombinations of these domains determine the stiffness of titin and thusthe stiffness of muscle tissue. Additional sarcomeric protein bindingsites have been found on titin, confirming the key role that titin playsin the assembly of sarcomere units (Gautel M. et al., J. Cell Science,109:2747-2754, 1996).

Monoclonal antibodies specific to titin have shown that single titinmolecules extend across the entire distance from the Z-disk to theM-line and consequently span in vivo a distance of more than onemicrometer. In its I-band section, the titin filament behaveselastically during muscle contraction, and thus is believed to accountfor most of the resting tension of striated muscle.

In addition to elastic elements contained in titin, there also areseveral titin-specific sequences and one kinase domain, although theexact role of these titin-specific sequences with respect totitin-related functions are not well understood.

Titin also is expressed in non-muscle tissues and cells (Banes, A. etal., Osteoarthritis and Cartilage, 7:141-153, 1999), and severalisoforms of titin with quite different sizes have been reported (Wang,K. et al., Proc. Natl. Acad. Sci., USA, 88:7101-5, 1991), suggestingthat titin may not be muscle-specific but rather may work as a universalelastic protein in tissues and organelles.

Analysis of the complete sequence of titin reveals that there areseveral titin-specific sequence insertions at its amino and carboxyltermini, with the exception of the Ig and FN III domains. Using thePSORT II program of Nakai and Horton (Trends Biochem. Sci., 24:34-36,1999), a seven amino acid (amino acids 200-206) NLS sequence, PAKKTKT(SEQ ID NO: 4) was predicted. With the use of an enhanced greenfluorescence protein (EGFP) reporter system, immunostaining and confocalmicroscopy techniques, the motif of 200-PAKKTKT-206 was shown to be afunctional NLS which directed the N-terminus of titin and EGFP into thenucleus in different cell lines, including human osteoblast-like MG63,BHK21, MC3T3-E1 and COS-7 cells, therefore showing for the first timethat there is a functional NLS in human titin protein and substantiatingevidence that titin may be transported into the nucleus of cells.

Experimental Procedures

Antibodies

Anti-titin antibodies (Z1Z2 recognizes the N-terminus of titin, M8M9recognizes the C-terminus of titin) were received from the University ofMannheim, Germany. ALEXAFLUOR® 568 conjugated goat anti-rabbit IgG (H+L)was obtained from Molecular Probes (Eugene, Oreg.).

Primers

All primers used in the study were synthesized by MWG Biotech, Inc.(High Point, N.C.).

Prediction of Nuclear Localization Signal (NLS) Sequences

The potential NLS sequences in the N- and C-termini of human titinprotein were predicted using three web-based programs: NucPred,PredictNLS and the PSORT II program (Cokol, M. et al., EMBO Rep.,1:411-415, 2000; Nair, R. et al., Proteins, 53:917-930, 2003; Nakai, K.et al., Trends Biochem. Sci., 24:34-36, 1999).

Molecular Cloning

The N-terminal and C-terminal fragments of human titin were amplifiedand cloned into pcDNA3.1 (Invitrogen, Carlsbad, Calif.) and pEGFP (BDBiosciences Clontech, Mountain View, Calif.) vectors, respectively. TheN-terminus was comprised of residues 1-790, including domains Z1, Z2 andZ repeats. The C-terminus was comprised of residues 33,791-34,350,including domains M7-10.

For pcDNA3.1 constructs, the N- and C-terminal fragments of the humansequence were amplified using primers: N-terminus, 5′-AAA AGG ATC CCTATG ACA ACT CAA GCA CCG ACG TTT-3′ (forward) (SEQ ID NO: 5) and 5′-AAAACT CGA GAA TTA CTG TGA TGA TAT GTG CAT TCC CTT-3′(reverse) (SEQ ID NO:6); C-terminus, 5′-AAA AGG ATC CCT ATG TCT TCA GAC AGT GTT GCT AAATTT-3′ (forward) (SEQ ID NO: 7) and 5′-AAA ACT CGA GAA TTA AAT GGA TCGAAT ATG TAT ATT CAC-3′ (reverse) (SEQ ID NO: 8). For pEGFP constructs,the primers were: N-terminus, 5′-AAA ACT CGA GCT ATG ACA ACT CAA GCA CCGACG TTT-3′ (forward) (SEQ ID NO: 9) and 5′-AAA AGG ATC CAA CTG TGA TGATAT GTG CAT TCC CTT-3′ (reverse) (SEQ ID NO: 10); C-terminus, 5′-AAA ACTCGA GCT ATG TCT TCA GAC AGT GTT GCT AAA TTT-3′ (forward) (SEQ ID NO: 11)and 5′-AAA AGG ATC CAA AAT GGA TCG AAT ATG TAT ATT CAC AGT-3′ (reverse)(SEQ ID NO: 12).

In the constructs of the C-terminus, a start codon was added at the 5′end. In the pcDNA3.1 constructs, a stop codon (TAA) was added at the endof both N- and C-terminal fragments of titin due to the lack of stopcodons in the pcDNA3.1 plasmid. The fragments of titin were amplifiedfrom human skeletal muscle total RNA (Ambion, Austin, Tex., #7982) andcloned into pcDNA3.1 at BamH I-Xho sites or pEGFP-C1 and N1 at XhoI-BamH I sites. NLS-localizing constructs, also referred to as nuclearfragments (NFs) 1-7 were cloned in pEGFP-C1 at Xho I-BamH I sites. NFs8-10 were cloned in pEGFP-C1 at Nhe I-Hind III sites. The primers usedfor making these NF constructs are shown in Table 1. To reduce theeffects of the C-terminal lysine residue of GFP on the nuclearlocalization of NFs 8-10, it was mutated to serine in these threeconstructs (shown in Table 1).

Transfection of Mammalian Cells

The constructs were transformed into the DH5α strain of E. coli(Invitrogen, Carlsbad, Calif.) and the transformed bacteria wereselected on LB selective medium containing 50 μg/ml kanamycin (Sigma St.Louis, Mo.). The construct plasmids were purified from DH5α using theplasmid Maxi kit from QIAGEN (Valencia, Calif.). The pEGFP or pcDNA3.1constructs were transfected into MC3T3-E1 cells (a mouse osteoblast-likecell line), BHK21 cells (derived from hamster kidney cells), COS-7(derived from monkey kidney cells) or MG63 cells (obtained from a humanosteosarcoma) using lipofectamine (Invitrogen, Carlsbad, Calif.)according to the manufacturer's protocol. The stable transfectants ofMG63 were selected using G418 (Invitrogen, Carlsbad, Calif.) 48 hourspost-transfection at 500 μg/ml. The cells were incubated with G418 forup to three weeks until individual colonies were formed. The colonieswith green fluorescence were selected using an Olympus BH61 fluorescencemicroscope. The colonies expressing N- or C-terminal fragments of titinwere selected by immunostaining. The stable transfectants of MG63 werekept in MEM medium (Invitrogen, Carlsbad, Calif.) containing 100 μg/mlG418.

Immunostaining of the N-Terminus and C-Terminus of Titin Expressed inCOS-7 Cells

N-terminal and C-terminal pcDNA3.1 titin constructs were transfectedinto COS-7 cells. The transiently transfected COS-7 cells were fixedwith 3.7% formaldehyde at room temperature for 30 minutes andpermeabilized with 0.1% Triton X-100 at room temperature for 15 minutes.After washing with phosphate-buffered saline (PBS, Invitrogen, Carlsbad,Calif.), the cells were blocked with 5% bovine serum albumin (BSA,Fisher Scientific, Suwanee, Ga.) and 2% goat serum (Sigma, St. Louis,Mo.) at 37° C. for 2 hours, then were labeled with anti-titin antibodyZ1Z2 or M8M9 (1:10 diluted in PBS) at 37° C. for 2 hours, then washedwith PBS two times, 5 minutes per wash. The proteins were visualizedwith ALEXAFLUOR® 568-conjugated goat, anti-rabbit IgG (1:500 diluted inPBS) at 37° C. for 1 hour. The stained cells were mounted on glassslides using a SLOWFADE® light antifade kit (Molecular Probes, Eugene,Oreg.) containing 100 ng/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma,St. Louis, Mo.). The images of the cells were viewed using a regularfluorescence microscope (Olympus BX60, OPELCO, Dulles, Va.) or aLeicaSP2 AOBS laser scanning confocal microscope (Leica Microsystem,Inc., Exton, Pa.) with a 40× oil immersion objective.

Prediction of Nuclear Export Signals (NES) Sequences

The amino acid sequences of the domains containing titin-specificsequences were retrieved from Genbank and input to a web-based NESprediction program, Net NES 1.1 (La Cour et al., Protein Eng. Des. Sel.,17:527-536, 2004). The amino acid residue numbering was derived from thecorresponding entries and was not the same for all the domains due tothe use of different entries.

Results

Nuclear Localization of Titin N-Terminal Fragment-GFP Fusion Proteins inMammalian Cells

In all of the tested cell lines, the titin N-terminal fusion proteinslocalized principally in the nucleus in both high-expressing andlow-expressing cells, while the titin C-terminal GFP fusion proteins andGFP only control proteins distributed in both the nucleus and thecytoplasm (FIG. 1). To confirm that the intracellular localization ofthe N- and C-terminal fragments of human titin protein was not due tothe effects of GFP fusion, the tag-free fragments were also cloned intopcDNA3.1 and transfected into COS-7 cells. The transiently expressedtitin fragments in COS-7 cells were stained with anti-titin antibodies.The results confirmed the finding of GFP fusion constructs. As shown inFIG. 2, the N-terminal fragment of titin mainly was in the nucleus whilethe C-terminal fragment of titin was mainly in the cytoplasm. Asconfirmed by DAPI staining, the titin N-terminal fragments clearly werein the nucleus and not in the nucleolus.

Prediction of Potential Nuclear Localization Signals in Amino andCarboxyl Termini of Human Titin

The sequences of titin N- and C-termini were input to three web-basedprograms as described in the experimental protocol section. No NLSsequences were predicted within the C-terminus of human titin from anyof the programs. Negative results on the N-terminus were obtained fromprograms PredictNLS and NucPred. One potential NLS sequence within theN-terminus of human titin was predicted by PSORT II: 200-PAKKTKT-206(pat7) (SEQ ID NO: 4) (Nakai K. et al., Trends Biochem. Sci., 24:34-36,1999). There were three types of NLS sequences based on theclassification of the PSORT II program. Pat4 was composed of four basicamino acids (K or R), or composed of three basic amino acids (K or R)and either H or P. Pat7 was a pattern of NLS sequences starting with Pand followed within 3 residues by a basic segment containing 3 of 4 K/Rresidues. It has been shown that bipartite NLS sequences are composed of2 basic residues, a 10-residue spacer and another basic regionconsisting of at least 3 of 5 basic residues (Nakai K. et al., TrendsBiochem. Sci., 24:34-36, 1999; Robbins, J. et al., Cell, 64:615-623,1991). The predicted potential NLS sequence was located in thetitin-specific sequence between the Z2 domain and Z repeats (FIG. 3).

By using the PSORT II program, three potential DNA-binding motifs werepredicted: 3460-LSAEEEGLHSAELQLSKINETL-3481 (SEQ ID NO: 13),11047-LPEEEEVLPEEEEVLPEEEEVL-11068 (SEQ ID NO: 14), and33195-LLRRRRSLSPTYIELMRPVSEL-33218 (SEQ ID NO: 15).

PAKKTKT is a Functional NES Sequence

Only one potential NLS sequence was predicted by the PSORT II program.However, it was thought that there may be other non-classic NLSsequences in the titin-specific region at the N-terminus of human titinprotein and, moreover, other domains may affect the function of this NLSsequence. Thus, to investigate the potential NLS sequences within theN-terminus of titin, eleven constructs were made in the enhanced greenfluorescence reporter systems: pEGFP-C1 and pEGFP-N1 (FIG. 4). Theseconstructs were transiently expressed in COS-7 cells. EGFP alone, as acontrol, showed both cytoplasmic and nuclear localization. The titin Cterminus linked to the N- or C-terminus of EGFP was distributed in boththe cytoplasm and nucleus. The N terminus linked to the N- or C-terminusof EGFP showed only nuclear localization, which indicated that theremight be potential nuclear localization signals within the N-terminus oftitin. The intracellular localization of constructs NF 1-5 showed that apotential NLS sequence localized between residues 180 and 209. Furtherdeletion of residues in constructs NF 6-11 minimized the NLS sequence toa seven amino acid sequence: residues 200 to 206. The deletion ofresidues proline and alanine almost ablated the NLS function. Theseconstructs also were transfected into MG63 cells and the stabletransfectants for each construct were selected, in which the resultswere the same as what was observed in COS-7 cells (FIG. 5).

Nuclear Export Signal (NES) Sequences in Human Titin

A total of six potential NES sequences were predicted by the NetNES 1.1program, as follows: 1705-FKKKLTSLRL-1714 (NES1) (SEQ ID NO: 16) in theN-terminus; the NES localized in Z repeats at the boundary of lowcomplexity and an Ig domain; 1077-MALMLIV-1083 (NES2) (SEQ ID NO: 17) inNovex I, which localized within an Ig domain; both 3900-IKKDDLRELGL-3910(NES3) (SEQ ID NO: 18) and 4712-LDILKTDLSL-4721 (NES4) (SEQ ID NO: 19)in Novex III localized in the extended region between Ig domains withina titin-specific sequence; 8923-LTTKEIKLEL-8932 (NES5) (SEQ ID NO: 20)localized to a low complexity, titin-specific sequence at the carboxyterminus of N2A; and 33040-LRLEEELEL-33048 (NES6) (SEQ ID NO: 21)localized in the low complexity, titin-specific sequence between M3 andM4 domains. (The conserved hydrophobic residues are underlined). Themost important properties of the NES sequences have been shown to beaccessibility and flexibility, which allow for receptor proteins tointeract with the signals (La Cour et al., Protein Eng. Des. Sel.,17:527-536, 2004). Therefore, NES sequences 1 and 2 are less likely tobe functional NES sequences.

Discussion

Titin is a giant filamentous protein, highly expressed in striatedmuscle tissues (the third most highly expressed protein after actin andmyosin in muscle), mainly composed of Ig, Fn III and PEVK repeats. Titinplays an important role in the assembly of the sarcomere and contributesto the elasticity of muscle tissues. A deficiency in titin proteinresults in severe heart or skeletal muscle diseases. Hence, titin is animportant architectural and regulatory protein. However, the functionsof the titin-specific sequences at the amino and carboxyl termini oftitin have not been explored. It has been argued that titin is foundprincipally in the cytoplasm bound with myosin. Recent studies, however,indicate that titin may not only appear in the cytoplasm but also in thenucleus (Machado, C. et al., J. Cell Biol., 151:639-652, 2000). Nucleartitin may play important roles in regulating chromosome condensation andspindle organization (Wernyj, R. et al., Cell Motil. Cytoskeleton,50:101-113, 2001). Due to its huge size, it is not feasible to determineits nuclear localization by heterogeneous expression of the wholeprotein. Analysis of the complete sequence of human titin revealed thatthere are several titin-specific sequence insertions at both the N- andC-termini (Bang, M. et al., Circ. Res., 89:1065-1072, 2001). However,there is no report of potential NLS sequences in human titin. Thisinvestigation constructed and transiently expressed GFP-titin fusionproteins of N- and C-terminal fragments in several mammalian cell linesand, for the first time, demonstrated that there is a functional NLSwithin the titin-specific region at the amino terminus of human titin,which indirectly supports the finding that titin occurs in the nucleus.When P-A residues are deleted, the NLS is disrupted. The results fromthis study suggest that a proline residue may be important formaintaining the accessibility and flexibility of the NLS so that thesignals may be accessible to the receptor proteins.

The mechanism for regulating the importation of classic NLS-containingnuclear proteins has been well investigated (Adam, S. et al., Cell,66:837-847, 1991; Gorlich, D., EMBO J., 17:2721-2727, 1998; Imamoto, N.et al., EMBO J., 14:3617-3626, 1995; Moroianu, J. et al., Proc. Natl.Acad. Sci. USA, 92:2008-2011, 1995). The first step is thereorganization of NLS sequences by importin α. This step can beregulated by the blockage of NLS sequences by mask domains or partnerproteins. Because titin is a filamentous protein and the NLS sequence(s)locates outside of Ig domains, it is likely that this step may beregulated by titin-binding proteins. Many titin N-terminus-bindingproteins have been reported, such as T-cap, obscuring, α-actinin andtelethonin (Gregorio, C. et al., Curr. Opin. Cell Biol., 11:18-25, 1999;McElhinny, A. et al., J. Cell Biol., 157:125-136, 2002; Pyle, W. G. etal., Circ. Res., 94:296-305, 2004; Sanger J. et al., J. Cell Biol.,154:21-24, 2001). The potential regulators for the importation of titininto the nucleus may be among them.

NES sequences were found in most of the shuttle proteins which can betransported into and exported out of the nucleus, such as actin, NF-κB,NF-AT and hnRNP (Harhaj, E. et al., Mol. Cell Biol., 19:7088-7095, 1999;Michael, W. et al., EMBO J, 16:3587-3598, 1997; Wada, A. et al., EMBO J,17:1635-1641, 1998; Zhu, J. et al., Nature, 398:256-260, 1999). Becausetitin appears to be transported to the nucleus as a potential shuttleprotein, then there also may be NES sequences in titin. As expected,several NES sequences were predicted in the N-terminus: Novex I and III,N2A; and in the C-terminus by the program NetNES 1.1 (La Cour et al.,Protein Eng. Des. Sel., 17:527-536, 2004). Many of the reported shuttleproteins are known to be involved in signal transduction/gene regulationand cell cycle regulation, which suggest that titin may play multipleroles other than only as an elastic protein. It has been shown that thekinase domain of titin regulates the expression of muscle genes (Lange,S. et al., Science, 308:1599-1603, 2005).

In conclusion, the results from this investigation indicate that titinappears to be a shuttle protein which is localized both in the cytoplasmand in the nucleus of cells. Thus, titin not only plays a cytoskeletalrole but also apparently participates in the regulation of genetranscription and mitosis by regulating chromosomal configuration andspindle contraction in the nucleus.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims. TABLE 1 Primers formaking the NLS-localizing constructs in pEGFP-C1 Clone Forward (5′ to3′) Reverse (5′ to 3′) NF-1 AAAACTCGAGCTATGACAACTCAAAAAAGGATCCAACTTCATTATTGCTTCTTG GCA CCG ACG TTT AGT TAC NF-2AAAACTCGAGCTATGACAACTCAA AAAAGGATCCAAAGTAATTTCTTCAGAAAT GCA CCG ACG TTTTCTAGT NF-3 AAAACTCGAGCTATGACAAGTCAA AAAAGGATCCAATCGGGTTTGTCTTGATTC GCACCG ACG TTT TGAGAT NF-4 AAAACTCGAGCTATGACAACTCAAAAAAGGATCCAAATTGGTGGCATTTACTGA GCA CCG ACG TTT ATAGGT NF-5AAAACTCGAGCTTCGACTGCTCAG AAAAGGATCCAACTGTGATGATATGTGCAT ATC TCA GAATCCCTT NF-6 AAAACTCGAGCTGGCCGCGCTAAA AAAAGGATCCAATCGGGTTTGTCTTGATTC CTGACG ATC TGAGAT NF-7 AAAACTCGAGCTGTGAGACTCCAAAAAAGGATCCAATCGGGTTTGTCTTGATTC GTG AGA GTG ACT TGAGAT NF-8AAAACTCGAGCTAGCGTTGGAAGA AAAAGGATCCAATCGGGTTTGTCTTGATTC GCT ACT TCG ACTTGAGAT NF-9 TCCGCTAGCGCTACCGGTCGCCAC AAAAAGCTTTTAAACAATTGTCTTTGTCTTTTTAGCAGGTACTTCTTCTTCACCTTGAAC CTTGTACAGCTCGTCCATGCCGA NF-10TCCGCTAGCGCTACCGGTCGCCAC AAAAAGCTTTTAAACAATTGTCTTTGTCTTTTTAGCAGGTACCTTGTACAGCTCGTCCAT GCC GA NF-11 TCCGCTAGCGCTACCGGTCGCCACAAAAAGCTTTTATGTCTTTGTCTTTTTAGC AGGGCTGTACAGCTCGTCCATGCCGA NF-12TCCGCTAGCGCTACCGGTCGCCAC AAAAAGCTTTTATGTCTTTGTCTTTTTGCTGTACAGCTCGTCCATGCCGANote: The C-terminal residue of EGFP was mutated from lysine to serine(AAG to AGC) in NFs 11-12.

TABLE 2 Prediction of Nuclear Export Signals (NES) in Human Titin.Domains Predicted NES N-terminus 1705-FKKKLTSLRL-1714 Novex I1077-MALMLIV-1083 Novex II None Novex III 3900-IKKDDLRELGL-39104712-LDILKTDLSL-4721 N2B None N2A 8923-LTTKEIKLEL-8932 C-terminus33040-LRLEEELEL-33048Note:The amino acid residue numbers were from the Genbank entries as follows:N-terminus (CAD12456), Novex I (CAD12459.1), Novex II (CAD12458.1),Novex III (NP_596870.1), N2B (CAD12455), N2A (NP_596869) and C-terminus(CAD12456). The conserved hydrophobic residues were underlined.

1. A nuclear localization signaling sequence derived from titin,consisting of amino acids as contained in SEQ ID NO: 1 and fragmentsthereof, wherein said nuclear localization signaling sequence is linkedto an agent in order to transport the agent into the nucleus of a cell.2. The nuclear localization signaling sequence according to claim 1,wherein said agent is linked to an antibody or ligand to form anNLS-agent-antibody or NLS-agent-ligand complex, in which said complexrecognizes a specific cell surface-expressing antigen or receptor,respectively, on a cell in order to enter the cytoplasm of cell beforebeing transported into the nucleus of the cell.
 3. The nuclearlocalization signaling sequence according to claim 1, wherein said agentis comprised of one or more peptides, proteins or nucleotides.
 4. Thenuclear localization signaling sequence according to claim 1, whereinsaid fragments of said SEQ ID NO: 1 is selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:
 4. 5. Thenuclear localization signaling sequence according to claim 1, whereinone fragment of said SEQ ID NO: 1 is comprised of SEQ ID NO:
 4. 6. Thenuclear localization signaling sequence according to claim 1, whereinthe cell is a mammalian cell.
 7. The nuclear localization signalingsequence according to claim 6, wherein the mammalian cell is a humancell.
 8. A method for transporting target proteins into the nucleus ofcells, comprising combining a cDNA containing the NLS sequence orfragments thereof according to claim 1 with a nucleic acid sequence forthe target protein to form a NLS-nucleic acid construct, wherein theexpressed protein of the cDNA-nucleic acid construct is capable ofentering the nucleus of cells to perform one or more specific functions.9. The method according to claim 8, wherein the one or more specificfunctions are selected from the group consisting of protecting thenucleus from toxic chemicals, radiation or other DNA-modifying agents;regulation of transcription, development or differentiation; inductionof DNA arrest (blockade); apoptosis (cell death) or DNA synthesis;delivery of anti-cancer agents; and any procedure where an agent islocalized to the nucleus of a target cell for any purpose.
 10. A kit,comprising the nuclear localization signal (NLS) sequences or fragmentsthereof according to claim 1 combined with a cDNA or RNA construct toform an NLS-cDNA or NLS-RNA construct.
 11. The kit according to claim10, wherein the NLS-cDNA or NLS-RNA construct is targeted to the nucleusof a cell and is incorporated into the genome of the cell, to beexpressed as a mRNA and ultimately translated into one or more proteins.12. The kit according to claim 10, wherein the NLS-cDNA or NLS-RNAconstruct transfers silencing RNA into the nucleus of a cell in order tointerfere with transcription of a native mRNA.